JP7425403B2 - Biological sample analysis method - Google Patents

Description

The present invention relates to a biological sample analysis method for analyzing the inclusions of exosomes contained in a biological sample and counting the number of exosomes.

By detecting and analyzing antigens associated with specific diseases as biomarkers, it is possible to discover specific diseases or verify the effectiveness of treatments for specific diseases. Exosomes, which are secreted from cells and contained in various body fluids such as blood, are expected to serve as biomarkers for discovering specific diseases.

Exosomes are minute membrane vesicles covered with a lipid bilayer membrane, and the lipid bilayer membrane contains various membrane proteins such as CD9 and CD63. It is known that when a person suffers from a specific disease, membrane proteins associated with the disease affect the amount of exosomes expressed on the surface of the lipid bilayer membrane (exosome surface). By using disease-related membrane proteins expressed on the surface of exosomes as antigens and measuring the number of exosomes that contain disease-related antigens, it is possible to discover whether a person is suffering from a specific disease. There is sex.

Inside the exosome, there are inclusions such as miRNA (microRNA), nucleic acids such as DNA, and proteins. Exosome inclusions are also expected to serve as biomarkers for discovering specific diseases (see Patent Document 1).

Japanese Patent Application Publication No. 2018-163043 JP 2017-40595 Publication JP2019-211366A

Conventionally, two biological samples containing exosomes are prepared, the inclusions of exosomes are analyzed in one sample, and the number of exosomes is measured in the other sample. That is, the exosomes whose inclusions were analyzed and the exosomes whose number was measured are not the same. It is desirable to analyze the inclusions of exosomes and count the number of exosomes in one sample.

An object of the present invention is to provide a biological sample analysis method that can analyze the inclusions of exosomes and measure the number of exosomes in one biological sample.

The present invention provides a reaction vessel in which a first buffer solution containing a plurality of first beads having a first antibody that specifically binds to a first antigen associated with a first disease immobilized thereon is injected. , a plurality of second beads having a second antibody that specifically binds to a second antigen associated with a second disease immobilized on the surface, and the first antigen and the second antigen on the surface. injecting a biological sample containing exosomes to be analyzed to generate a second buffer containing the exosomes to be analyzed to which the first beads and the second beads are bound; The exosome to be analyzed to which the first bead and the second bead are bound is collected from the solution, and the exosome to be analyzed to which the first bead and the second bead are bound to the collected exosome to be analyzed is recovered. The first bead is separated, the exosome to be analyzed to which the second bead is bound is collected, and the exosome to be analyzed to which the recovered second bead is bound is subjected to a lysis treatment, and the second bead is bound to the exosome to be analyzed. A biological sample analysis method comprising separating beads and inclusions of the exosomes to be analyzed, collecting each of them, analyzing the inclusions of the collected exosomes to be analyzed, and counting the number of recovered second beads. I will provide a.

According to the biological sample analysis method of the present invention, it is possible to analyze the inclusions of exosomes and measure the number of exosomes using one biological sample.

1 is a flowchart partially illustrating a biological sample analysis method of one embodiment. 1A is a flowchart partially illustrating a biological sample analysis method of one embodiment; FIG. FIG. 2 is a conceptual diagram showing magnetic microbeads used in a biological sample analysis method of one embodiment. FIG. 2 is a conceptual diagram showing a reaction container into which a buffer solution containing magnetic microbeads is injected. FIG. 2 is a conceptual diagram showing a biological sample specimen containing single-positive exosomes and double-positive exosomes. FIG. 2 is a conceptual diagram showing the structure of a single-positive exosome. FIG. 2 is a conceptual diagram showing the structure of a double-positive exosome. FIG. 2 is a conceptual diagram showing antibody nanobeads used in the biological sample analysis method of one embodiment. A conceptual diagram showing a state in which a sample containing double-positive exosomes and antibody nanobeads are injected into a reaction container containing a buffer solution containing magnetic microbeads, and double-positive exosomes are captured in a sandwich between magnetic microbeads and antibody nanobeads. be. FIG. 8 is a conceptual diagram showing a state in which unnecessary substances other than magnetic microbeads and antibody nanobeads in which double-positive exosomes are sandwich-captured are removed from the reaction container shown in FIG. 7. 8 is a diagram showing a method for separating unnecessary substances from the reaction container shown in FIG. 7. FIG. FIG. 2 is a conceptual diagram showing a state in which exosomes are separated from magnetic microbeads in magnetic microbeads and antibody nanobeads in which double-positive exosomes are sandwich-captured. FIG. 2 is a diagram showing a method for separating exosomes from magnetic microbeads using haptens. FIG. 3 is a diagram showing a method of separating exosomes from magnetic microbeads using a linker. FIG. 2 is a conceptual diagram showing magnetic microbeads obtained by separating magnetic microbeads, antibody nanobeads capturing double-positive exosomes, and single-positive exosomes. FIG. 2 is a conceptual diagram showing antibody nanobeads that capture double-positive exosomes and single-positive exosomes obtained by separating magnetic microbeads, antibody nanobeads that capture double-positive exosomes, and single-positive exosomes. FIG. 3 is a diagram showing the middle steps of a method for separating magnetic microbeads and antibody nanobeads. FIG. 15A is a diagram showing the steps following FIG. 15A in a method for separating magnetic microbeads and antibody nanobeads. FIG. 2 is a conceptual diagram showing antibody nanobeads that capture double-positive exosomes, obtained by separating antibody nanobeads that capture double-positive exosomes from single-positive exosomes. FIG. 2 is a conceptual diagram showing single-positive exosomes obtained by separating antibody nanobeads that capture double-positive exosomes from single-positive exosomes. FIG. 2 is a conceptual diagram showing a state in which antibody nanobeads are separated from double-positive exosomes. FIG. 2 is a conceptual diagram showing antibody nanobeads obtained by separating antibody nanobeads and double-positive exosomes that have been separated from each other. FIG. 2 is a conceptual diagram showing double-positive exosomes obtained by separating antibody nanobeads and double-positive exosomes that have been separated from each other. 1B is a flowchart showing a specific method for measuring the number of antibody nanobeads in step S122 of FIG. 1B. FIG. 3 is a diagram showing a method for measuring the number of antibody nanobeads. FIG. 2 is a conceptual diagram showing a state in which polar functional groups for attaching antibody nanobeads to a disk substrate are formed.

A biological sample analysis method according to one embodiment will be described below with reference to the accompanying drawings. In this embodiment, it is possible to capture double-positive exosomes in which two types of specific membrane proteins are expressed on the surface of one exosome, analyze the inclusions, and measure the number of exosomes. Let us take a biological sample analysis method as an example.

In FIG. 1A, in step S11, the operator immobilizes a plurality of antibodies 11 that specifically bind to the antigen associated with the first disease on the surface of magnetic microbeads 10, as shown in FIG. As shown in FIG. 3, in step S12, the operator injects a buffer solution 21 (first buffer solution) containing a large number of magnetic microbeads 10 on which antibodies 11 are immobilized into the reaction container 20. The diameter of the magnetic microbeads 10 is approximately 3 μm, and the diameter may be on the order of micrometers.

As shown in FIG. 4, the operator prepares a biological sample specimen 31 in a container 30. As shown in FIG. The specimen 31 includes a single-positive exosome 32s and a double-positive exosome 32w, which will be described later. The specimen 31 is any body fluid such as blood, and contains impurities 33 and 34, which are unnecessary substances, in addition to exosomes 32s and 32w. Single-positive exosomes 32s and double-positive exosomes 32w are collectively referred to as exosomes 32.

As conceptually shown in FIG. 5A, the single-positive exosome 32s is covered with a lipid bilayer membrane 320, and membrane proteins 321 to 323 are present in the lipid bilayer membrane 320. Membrane protein 321 is CD9, membrane protein 322 is CD63, and membrane protein 323 is CD147. CD9 and CD63 are normally present membrane proteins, and CD147 is an example of a membrane protein associated with the first disease. In this way, a single positive exosome 32s refers to an exosome 32 in which one type of disease-related membrane protein (CD147 in this embodiment) is present on the surface of one exosome 32.

As conceptually shown in FIG. 5B, the double-positive exosome 32w is covered with a lipid bilayer membrane 320, and membrane proteins 321 to 324 are present in the lipid bilayer membrane 320. Membrane protein 324 is HER2 (CD340). HER2 (CD340) is an example of a membrane protein associated with a second disease. In this way, double-positive exosomes 32w are exosomes 32 in which two types of disease-related membrane proteins (CD147 and HER2 (CD340) in this embodiment) are present on the surface of one exosome 32. Point. Hereinafter, HER2 (CD340) will be referred to as HER2, which is a common name.

In this embodiment, as an example, a double-positive exosome 32w in which both CD147 and HER2 are present on the surface is the exosome to be analyzed. In this embodiment, CD147 is used as the first antigen associated with the first disease, and HER2 is used as the second antigen associated with the second disease, but the present invention is not limited thereto. Antibody 11 immobilized on the surface of magnetic microbeads 10 is a first antibody that specifically binds to CD147.

Inside the exosome 32, there are inclusions such as nucleic acids such as miRNA 325 and DNA 326, and proteins (not shown).

As shown in FIG. 6, the operator prepares in a container 40 a buffer solution 41 containing antibody nanobeads 50 with a plurality of antibodies 51 immobilized on the surface. The antibody nanobeads 50 are much smaller in diameter than the magnetic microbeads 10, with a diameter of about 200 nm. The diameter of the antibody nanobeads 50 may be less than 1 μm, preferably 100 nm to 500 nm. The antibody 51 immobilized on the surface of the antibody nanobeads 50 is an antibody (second antibody) that specifically binds to HER2, which is a second antigen associated with the second disease.

As shown in FIG. 7, the operator sequentially (or simultaneously) injects the specimen 31 containing the exosomes 32 and the buffer solution 41 containing the antibody nanobeads 50 into the reaction container 20 in step S13. When the buffer solution 21 is a mixture of the buffer solution 21 and the buffer solution 41, the reaction container 20 contains a buffer solution containing double-positive exosomes 32w sandwiched between the magnetic microbeads 10 and the antibody nanobeads 50. 21 (second buffer) is produced. In this case, since sandwich capture means that two beads bind to one exosome, a second buffer containing exosomes 32w to which magnetic microbeads 10 and antibody nanobeads 50 are bound is generated. It can be said that At this time, the operator shakes the reaction container 20 as necessary and waits for a predetermined time.

In FIG. 7, double-positive exosomes 32w, in which both CD147 and HER2 are present on the surface, are sandwich-captured with magnetic microbeads 10 and antibody nanobeads 50. Single positive exosome 32s in which HER2 is not present binds to antibody 11 of magnetic microbeads 10. However, since the antibody nanobeads 50 do not bind to the single-positive exosomes 32s, the single-positive exosomes 32s are not sandwich-captured by the magnetic microbeads 10 and the antibody nanobeads 50.

In FIG. 7, a large number of magnetic microbeads 10 are present in the reaction container 20, but for ease of understanding, only one magnetic microbead 10 is shown in an enlarged manner.

In step S14, the operator separates the magnetic microbeads 10 to which the antibody nanobeads 50 are linked via the exosomes 32 from unnecessary materials by magnetic collection, weak centrifugation, or the like. The operator washes the separated magnetic microbeads 10. Antibody nanobeads 50 and contaminants 33 and 34 that are not linked to magnetic microbeads 10 are unnecessary. Note that since the antibody nanobeads 50 are injected in excess in step S13, the unnecessary substances are mainly the antibody nanobeads 50 that are not linked to the magnetic microbeads 10. As a result of step S14, the magnetic microbeads 10 shown in FIG. 8 to which the antibody nanobeads 50 are linked via the exosomes 32 can be recovered.

Exosomes 32s to which antibody nanobeads 50 are not bound are also bound to the recovered magnetic microbeads 10. Of course, among the plurality of magnetic microbeads 10, there are magnetic microbeads 10 to which only exosomes 32w to which antibody nanobeads 50 are bound are bound, and there are magnetic microbeads 10 to which only exosomes 32s to which antibody nanobeads 50 are not bound are bound. There are also magnetic microbeads 10.

By the way, if the antibody nanobeads 50 are not magnetic nanobeads, the magnetic microbeads 10 and the antibody nanobeads 50 not linked to the magnetic microbeads 10 can be easily separated by magnetically capturing the magnetic microbeads 10. can. When counting the number of antibody nanobeads 50 linked to the magnetic microbeads 10 in step S22, which will be described later, the antibody nanobeads 50 are preferably magnetic nanobeads. Even if the antibody nanobeads 50 are magnetic nanobeads, it is possible to separate the magnetic microbeads 10 from the unbound antibody nanobeads 50.

Specifically, as shown in FIG. 9, magnetic microbeads 10 and unbound antibody nanobeads 50 can be separated. In FIG. 9, (a) shows a state in which a buffer solution 21 containing magnetic microbeads 10 to which antibody nanobeads 50 are linked and antibody nanobeads 50 that are not linked is injected into a container 60. FIG. 9(a) corresponds to the state shown in FIG. By magnetic collection or weak centrifugation using a magnet placed at the bottom of the container 60, the magnetic microbeads 10 and the unbound antibody nanobeads 50 can be largely separated, as shown in FIG. 9(b).

This is because the volumes of the magnetic microbeads 10 and the antibody nanobeads 50 are about 1000 times different, so the magnetic microbeads 10 are magnetically captured in a short time, whereas the antibody nanobeads 50 are magnetically captured only for a long time. This is because it is not done. By utilizing this time difference in magnetic capture, the magnetic microbeads 10 and unbound antibody nanobeads 50 can be largely separated.

In the step of washing the magnetic microbeads 10, if the precipitate, which is mainly the magnetic microbeads 10 in FIG. 9(b), is collected, the supernatant is removed, and the buffer solution 21 is added, the result will be as shown in FIG. 9(c). As shown, unbound antibody nanobeads 50 can be significantly reduced. If magnetic collection is performed in the state shown in FIG. 9(c), the result will be as shown in FIG. 9(d), and if the precipitate is collected, the supernatant is removed, and buffer 21 is added, the result will be as shown in FIG. 9(e). In this way, the number of unbound antibody nanobeads 50 can be further significantly reduced.

If magnetic collection is performed in the state shown in FIG. 9(e), the result will be as shown in FIG. 9(f), and if the precipitate is collected, the supernatant is removed, and buffer 21 is added, the result will be as shown in FIG. 9(g). Thus, only the magnetic microbeads 10 to which the antibody nanobeads 50 are linked can be separated. (g) in FIG. 9 corresponds to the state in FIG. The number of times of precipitate collection and supernatant removal is not limited to the number of times shown in FIG.

As shown in FIG. 10, the operator performs a process of separating the exosomes 32 from the magnetic microbeads 10 in step S15. Then, the double-positive exosomes 32w to which the antibody nanobeads 50 are bound and the single-positive exosomes 32s to which the antibody nanobeads 50 are not bound are separated from the magnetic microbeads 10.

As the process of separating the exosomes 32 from the magnetic microbeads 10 in step S15, any of the following methods can be adopted. As a first method, there is a method using a hapten shown in FIG. A hapten is a substance that binds to antibodies, but because of its small molecular weight, does not exhibit the activity of inducing antibody production (immunogenicity) by itself. Haptens, when combined with appropriate proteins, become complete antigens with immunogenic properties.

As shown in FIG. 11, an anti-DNP antibody 52 is immobilized on the magnetic microbeads 10 instead of the antibody 51. DNP is 2,4-dinitrophenol and is an example of a hapten. A double-positive exosome 32w or a single-positive exosome 32s is bound to the anti-DNP antibody 52 via an antibody 54 bound to DNP53 (hereinafter referred to as DNP antibody 54). DNP antibody 54 is an antibody that specifically binds to CD147.

In this state, the binding between the anti-DNP antibody 52 and the DNP antibody 54 is an equilibrium reaction. When DNP53 (hapten) is added in large excess to buffer solution 21 (third buffer solution) containing exosomes 32w sandwich-captured with magnetic microbeads 10 and antibody nanobeads 50, DNP bound to anti-DNP antibody 52 is removed. The antibody 54 is replaced with DNP 53, and the magnetic microbeads 10 and the double-positive exosomes 32w and single-positive exosomes 32s bound to the antibody nanobeads 50 are separated.

A second method for separating exosomes 32 from magnetic microbeads 10 is to use a cleavable linker 55 shown in FIG. 12. Antibodies 11 are immobilized on the magnetic microbeads 10 via linkers 55. When using the second method, antibodies 11 are immobilized on magnetic microbeads 10 shown in FIG. 8 via linkers 55, and antibodies 11 are bound to exosomes 32.

When a linker cleavage reagent is added to the buffer solution 21 (third buffer solution) in the reaction container 20, the linker 55 is cleaved and the magnetic microbeads 10 and the antibody 11 are separated, as shown in FIG.

As a third method for separating the exosomes 32 from the magnetic microbeads 10, a Whole antibody may be used for the magnetic microbeads 10, and a Fab antibody may be used for the antibody nanobeads 50. By decomposing and digesting the whole antibody with an enzyme, the exosomes 32 can be separated from the magnetic microbeads 10.

In FIG. 1B, the operator separates the magnetic microbeads 10 from the antibody nanobeads 50 bound to the double-positive exosomes 32w and the single-positive exosomes 32s in step S16. By washing the separated magnetic microbeads 10, the magnetic microbeads 10 shown in FIG. 13 can be recovered. In FIG. 13, a buffer solution 21 containing only magnetic microbeads 10 is injected into a reaction container 20.

By washing the antibody nanobeads 50 and exosomes 32s bound to the separated exosomes 32w, the antibody nanobeads 50 and exosomes 32s bound to the exosomes 32w shown in FIG. 14 can be recovered. In FIG. 14, a buffer solution 21 containing exosomes 32s and antibody nanobeads 50 bound to exosomes 32w is injected into a container 22.

The separation of the magnetic microbeads 10 from the antibody nanobeads 50 bound to the exosomes 32w and the exosomes 32s in step S16 can be performed in the same manner as in FIG. 9. However, in the separation in step S16, it is necessary to collect the antibody nanobeads 50 and exosomes 32s bound to the exosomes 32w contained in the supernatant without discarding the supernatant.

Specifically, as shown in FIGS. 15A and 15B, the magnetic microbeads 10 and the antibody nanobeads 50 bound to the exosomes 32w can be separated. In FIGS. 15A and 15B, illustration of the exosome 32s is omitted.

In FIG. 15A, (a) shows a state in which a buffer solution 21 containing mutually separated magnetic microbeads 10 and antibody nanobeads 50 is injected into a container 61. (a) of FIG. 16A corresponds to the state of FIG. 10. As shown in FIG. 15A (b), the magnetic microbeads 10 and the antibody nanobeads 50 can be largely separated by magnetic collection or weak centrifugation using a magnet placed at the bottom of the container 61.

If the precipitate, which is mainly magnetic microbeads 10 in FIG. 15A (b), is collected and buffer solution 21 is added, the antibody nanobeads 50 can be significantly reduced as shown in FIG. 15A (c). As shown in FIG. 15A (d), by collecting the supernatant into a container 62, a buffer solution 21 containing a large amount of antibody nanobeads 50 can be obtained. If magnetic collection is performed in the state shown in FIG. 15A (c), the state shown in FIG. 15A (e) will be obtained.

If the precipitate, which is mainly magnetic microbeads 10 in FIG. 15A (e), is collected and buffer solution 21 is added, the antibody nanobeads 50 can be further reduced as shown in FIG. 15B (f). As shown in FIG. 15B (g), by collecting the supernatant into a container 63, a buffer solution 21 containing antibody nanobeads 50 can be obtained. If magnetic collection is performed in the state shown in FIG. 15B (f), the state shown in FIG. 15B (h) will be obtained.

If the precipitate containing only the magnetic microbeads 10 in (h) of FIG. 15B is collected and the buffer solution 21 is added, only the magnetic microbeads 10 without the antibody nanobeads 50 will be collected as shown in (i) of FIG. 15B. A buffer solution 21 containing the following can be obtained. (i) in FIG. 15B corresponds to the state in FIG. 13. As shown in FIG. 15B (j), by collecting the supernatant into a container 64, a buffer solution 21 containing a small amount of antibody nanobeads 50 can be obtained.

As shown in FIG. 15B (k), by combining all antibody nanobeads 50 from containers 62 to 64, all antibody nanobeads 50 bound to magnetic microbeads 10 and bound to exosomes 32w can be recovered. . At this time, exosomes 32s can also be collected. (k) in FIG. 15B corresponds to the state in FIG. 14. The number of times of precipitate collection and supernatant collection is not limited to the number of times shown in FIGS. 15A and 15B.

In FIG. 1B, in step S17, the operator separates the antibody nanobeads 50 bound to the exosomes 32w and the exosomes 32s. The antibody nanobeads 50 bound to the exosomes 32w and the exosomes 32s can be separated and individually collected by the same method as in FIGS. 15A and 15B.

By washing the antibody nanobeads 50 bound to the separated exosomes 32w, the antibody nanobeads 50 bound to the exosomes 32w shown in FIG. 16 can be recovered. In FIG. 16, a buffer solution 21 containing only antibody nanobeads 50 bound to exosomes 32w is injected into a container 23. By washing the separated exosomes 32s, the exosomes 32s shown in FIG. 17 can be recovered. In FIG. 17, a buffer solution 21 containing only exosomes 32s is injected into a container 24.

In step S18, the operator dissolves the exosomes 32s and analyzes the inclusions of the exosomes 32s. When a surfactant is added to the buffer solution 21 containing the exosomes 32s, the lipid bilayer membrane 320 of the exosomes 32s is dissolved and destroyed. In this embodiment, since the main purpose is to analyze double-positive exosomes 32w, step S18 may be omitted.

In step S19, the operator performs a process of separating the antibody nanobeads 50 and the exosomes 32w. As an example, by adding a surfactant to the buffer solution 21 shown in FIG. 16, the antibody nanobeads 50 and the exosomes 32w can be separated, as shown in FIG. 18. For example, the surfactant is a 2% solution of TritonX-100, a nonionic surfactant. Since the lipid bilayer membrane 320 of the exosome 32w is dissolved by the surfactant, the antibody nanobeads 50 and the exosome 32w are separated by destroying the exosome 32w, and the contents of the exosome 32w are dissolved in the buffer solution 21. is released. In FIG. 18, in reality, each exosome 32w has its lipid bilayer membrane 320 destroyed and is divided into a plurality of inclusions.

In step S20, the operator separates and collects the antibody nanobeads 50 and the inclusions of the exosomes 32w individually. The antibody nanobeads 50 and the inclusions of the exosomes 32w can be separated by the same method as in FIGS. 15A and 15B.

By washing the separated antibody nanobeads 50, the antibody nanobeads 50 shown in FIG. 19 can be recovered. In FIG. 19, a buffer solution 21 containing only antibody nanobeads 50 is injected into a container 24. By washing the separated contents of the exosomes 32w, the contents of the exosomes 32w shown in FIG. 20 can be recovered. Also in FIG. 20, each exosome 32w is actually divided into a plurality of inclusions. In FIG. 20, the buffer solution 21 containing only the contents of the exosomes 32w is injected into the container 25.

In step S21, the operator takes out the inclusions of the exosomes 32w in the container 25 and analyzes the inclusions. At this time, DNA or protein in the inclusions can be analyzed by analysis methods such as RT-PCR (Reverse Transcription - Polymerase Chain Reaction) analysis and LC-MS (Liquid Chromatography - Mass Spectrometry) analysis. Further, the operator counts the number of antibody nanobeads 50 in step S22. An operator may perform step S21 and step S22 in any order, and multiple operators may perform them simultaneously. The order of step S18, step S21, and step S22 is also arbitrary. When the operator executes steps S21 and S22 (or steps S18, S21, and S22), the biological sample analysis process ends.

Since the antibody nanobeads 50 whose number is counted in step S22 are bound to double-positive exosomes 32w, the number of exosomes 32w in which both CD147 and HER2 are present is counted in step S22. . The exosomes 32 whose inclusions are analyzed in step S21 are only double-positive exosomes 32w from which the single-positive exosomes 32s have been removed, and these are the exosomes 32w whose number is counted in step S22.

Therefore, the double-positive exosomes 32w whose inclusions were analyzed and the double-positive exosomes 32w whose number was measured are the same. In one specimen 31 shown in FIG. 4, it is possible to analyze the inclusions of double-positive exosomes 32w in which both disease-related membrane proteins CD147 and HER2 are present, and to measure the number of exosomes 32w. can.

A specific method for measuring the number of antibody nanobeads 50 in step S22 of FIG. 1B will be described using FIGS. 21 to 23. In FIG. 21, the operator, in step S221, as shown in FIG. Prepare 81. Antibody 82 is, for example, a Goat anti-mouse IgG antibody.

The disk substrate 81 is preferably disk-shaped, and preferably has concave portions 81G and convex portions 81L arranged alternately in the radial direction. The concave portion 81G and the convex portion 81L are formed in a spiral shape or a concentric shape.

As shown in FIG. 22(b), the operator attaches a cartridge 83 in which a plurality of, for example, cylindrical through holes are formed to the disk substrate 81. As shown in FIG. Then, a well 83W is formed by the disk substrate 81 and the cartridge 83. As shown in FIG. 22(c), the operator dispenses the buffer solution 21 containing the recovered antibody nanobeads 50 into each well 83W in step S222. Note that the cartridge 83 can adopt the configuration described in Patent Document 2.

As shown in FIG. 22(d), in step S223, the operator combines the antibody 51 of the antibody nanobeads 50 with the antibody 82 immobilized on the disk substrate 81 by shaking or magnetic adsorption. If the antibody nanobeads 50 are magnetic nanobeads and a magnet is placed on the back side of the disk substrate 81, the antibody nanobeads 50 will be attracted to the surface of the disk substrate 81. This makes it easier for the antibody 51 of the antibody nanobeads 50 to bind to the antibody 82 immobilized on the disk substrate 81.

The operator drains the supernatant in the well 83W in step S224, and dries the disk substrate 81 in step S225. As shown in FIG. 22(e), in step S226, the operator attaches the dried disk substrate 81 to the analyzer, and uses the analyzer to measure the number of antibody nanobeads 50 immobilized on the disk substrate 81. do. The analyzer measures the number of antibody nanobeads 50 by irradiating the disk substrate 81 with a laser beam focused by a condenser lens 101 . Note that the configuration described in Patent Document 3 can be adopted as the analyzer.

As shown in FIG. 23, instead of immobilizing the antibody 82 on the disk substrate 81, the surface of the disk substrate 81 may be treated with oxygen plasma to form a polar functional group such as a carboxyl group (COOH). The antibody 51 of the antibody nanobeads 50 is adsorbed to the polar functional group.

As described above, according to the biological sample analysis method of the present embodiment, one biological sample (specimen 31) can detect the inclusions of double-positive exosomes 32w containing two types of disease-related antigens (membrane proteins). The exosomes 32w can be analyzed and the number of exosomes 32w can be measured.

The present invention is not limited to the embodiment described above, and various modifications can be made without departing from the gist of the present invention. In this embodiment, the first beads are magnetic microbeads 10 and the second beads are antibody nanobeads 50, but the first beads and the second beads should be different in size to the extent that they can be separated from each other. good. If the first beads are microbeads and the second beads are nanobeads, they can be easily separated from each other, so it is preferable to use the first beads as microbeads and the second beads as nanobeads.

The first beads are preferably magnetic beads, but may also be non-magnetic beads. If the first beads are magnetic beads and the second beads are non-magnetic beads, they can be separated from each other extremely easily. If the second beads are magnetic beads, it becomes easier to fix the second beads to the disk substrate 81. The first beads may be non-magnetic beads and the second beads may be magnetic beads. The first beads and the second beads may be magnetic beads.

10 Magnetic microbeads 11 Antibodies 20 Reaction containers 21 Buffer solutions 22-25, 30, 40, 60, 61-65 Containers 31 Samples 32s Single positive exosomes 32w Double positive exosomes 51,82 Antibodies 52 Anti-DNP antibody 53 DNP
54 Antibody (DNP antibody)
55 Linker 81 Disk substrate 83 Cartridge 83W Well 101 Condenser lens 320 Lipid bilayer membrane 321-324 Membrane protein (antigen)
325 miRNA
326 DNA

Claims (5)

A second buffer solution is injected into a reaction vessel containing a first buffer solution containing a plurality of first beads on whose surface a first antibody that specifically binds to a first antigen associated with a first disease is immobilized. a plurality of second beads having a second antibody that specifically binds to a disease-related second antigen immobilized on the surface; and an exosome to be analyzed having the first antigen and the second antigen on the surface. injecting a biological sample containing the sample to generate a second buffer containing the exosome to be analyzed to which the first bead and the second bead are bound;
Recovering the analysis target exosome to which the first beads and the second beads are bound from the second buffer,
Separating the first beads from the exosomes to be analyzed to which the recovered first beads and the second beads are bound, and collecting the exosomes to be analyzed to which the second beads are bound;
Dissolving the exosomes to be analyzed to which the recovered second beads are bound, separating the second beads and the inclusions of the exosomes to be analyzed, and collecting each of them;
A biological sample analysis method, comprising analyzing the contents of the collected exosomes to be analyzed and counting the number of the collected second beads. the first antibody is immobilized on the first bead using a hapten,
The biological sample according to claim 1, wherein the first bead is separated by adding a hapten to a third buffer containing the exosome to be analyzed to which the first bead and the second bead are bound. Analysis method. the first antibody is immobilized to the first bead using a cleavable linker;
The first bead is separated by adding a cleavage reagent for the linker to a third buffer containing the exosome to be analyzed to which the first bead and the second bead are bound. The biological sample analysis method described. The biological sample analysis method according to any one of claims 1 to 3, wherein a surfactant is used in the dissolution treatment. The biological sample analysis method according to any one of claims 1 to 4, wherein the first beads are magnetic beads.

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